1. Field of the Invention
This invention relates to a device for cell culture production, and more particularly to a vessel and closure assembly having means for varying the gas diffusion rate into and out of the vessel.
2. Description of Related Art
Typically, cells are cultured under conditions in which the hydrogen ion concentration (pH, the negative logarithm of the hydrogen ion concentration), temperature, humidity, osmolarity and concentration of certain ions are controlled within relatively narrow limits.
Vessels that are used in such tissue culture systems are typically made from plastic and include closures. Such vessels and closures are illustrated in U.S. Pat. Nos. 4,289,248, 4,387,822, 4,770,308 and 5,047,347.
In typical tissue culture systems, pH is maintained near physiologic levels by a buffering system in the tissue culture fluid, in conjunction with an incubator in which carbon dioxide (CO.sub.2) is infused at a rate sufficient to maintain a concentration in the incubator atmosphere of approximately 5 to 7 volume percent. The CO.sub.2 reacts with water to form the weak acid, carbonic acid, which in turn interacts with the buffering system to maintain the pH near physiologic levels. Entry of CO.sub.2 from the incubator into the tissue culture vessel is generally achieved by utilizing a closure on the vessel such as, a loosely fitting cap, a stopper or a cap with a permeable membrane. Equilibrium in the vessel is maintained by allowing gas exchange with the inside of the vessel and the atmosphere of the incubator while preserving sterility and preventing liquid leakage. A loosely fitting cap or a stopper is partially opened to an approximate extent determined by the user or by the closure design.
Removal of the vessel from the controlled atmosphere of the incubator is often required during growth and culturing of cells. The vessels are usually removed for inspection and/or manipulation of the cells and culture fluids. It is important that the pH of the cell culture be maintained at the desired physiologic level while the vessel is outside of the incubator.
The limitations of the closures used with vessels for cell culturing such as those described in U.S. Pat. Nos. 4,289,248, 4,387,822, 4,770,308 and 5,047,347 are (1) susceptibility to contamination by microorganisms because of gas flow through the small but unobstructed space between cap and the vessel; (2) a slow and variable rate of achieving pH equilibration by diffusion of CO.sub.2 through the loose-fitting cap and (3) the rate of gas exchange can not be estimated by a loose fitting cap or a cover that can be moved continuously without limitation.
A special need exists for an improved closure for a culture vessel which: (1) provides rapid and uniform equilibration between the vessel atmosphere and the incubator; (2) allows the culture vessel to be removed from the controlled atmosphere of the incubator for reasonably long times without subjecting the cell culture to undesirable changes in the pH of the system; (3) eliminates the guesstimated procedure of increasing and/or decreasing the gas exchange rate in order to substantially regulate the concentration of CO.sub.2 inside the vessel; (4) provides a gas exchange rate that can be measured; and (5) provides precise control over the degree of venting.